The need for simple and efficient methods of labeling oligonucleotides has been present ever DNA synthesis became a routine laboratory procedure. Initially, practically all oligonucleotide labeling was radioactive in nature, involving a fairly straightforward enzymatic reaction consisting of adding a radioactive phosphate group from a nucleoside triphosphate, usually to the 5' end of the oligonucleotide.
However, the rapidly increasing cost of radioactive waste disposal together with an increased awareness of the potentially harmful effects of exposure to radiation have contributed to shifting the emphasis toward other ways of labeling synthetic oligonucleotides. In addition, the number of different applications where non-radioactively labeled oligonucleotides and nucleotides are being used has expanded significantly. Fluorescent In Situ Hybridization (FISH), sequencing, enzymatic amplification, and sandwich assays in microtiter plate format are but some of the applications where non-radioactively labeled oligonucleotides and nucleotides are useful.
Currently, nucleotides and synthetic oligonucleotides are generally labeled non-radioactively with two types of markers: "biological" moieties, such as biotin, Horse Radish Peroxidase (HRP), etc., and "chemical" moieties, such as fluorescent (e.g., Fluorescein, Rhodamine, etc.) or chemiluminescent (e.g. Lanthanides) groups, which have the advantage of being more readily detected.
The use of fluorescent labels with antibodies, DNA probes, biochemical analogs, lipids, drugs, cytokines, cells and polymers has expanded recent years. The wider use of fluorescent probes results partly from the evolution of advanced detection instrumentation, particularly electronic imaging microscopes and flow cytometry and partly from the availability of new fluorescent labeling reagents.
Cyanine and related dyes are "chemical" moieties, which have several desirable properties for use as sensitive detection labels. These dyes are strongly light absorbing and highly luminescent. They can be covalently attached to proteins and other biological and nonbiological markers to make these materials fluorescent so that they can be detected. The labeled materials can then be used in assays employing excitation light sources and luminescence detectors. Avidin labeled with cyanine type dyes can be used to quantify biotinylated materials and antibodies conjugated with cyanine-type dyes can be used to detect and measure antigens and haptens. Furthermore, cyanine-conjugated lectins can be used to detect specific carbohydrate groups. Moreover, cyanine-conjugated fragments of DNA or RNA can be used to identify the presence of complementary nucleotide sequences in DNA or RNA. The cyanine dyes have the advantage that by synthesizing structural modifications of the chromophore portion of the molecule, different fluorescent labeling reagents can be made that will absorb and fluoresce light at many different wavelengths in the visible and near infrared region of the spectrum. Also, the cyanine and related dyes have an advantage in that they can be synthesized with a variety of functional groups attached. This versatility permits control over such factors as the solubility of the dye and labeled product and helps reduce non-specific binding of the labeled material to irrelevant components in an assay mixture. This versatility also allows for selection of labeling reagents that minimally perturb the function of the labeled product.
Further desirable properties of these dyes include absorbance at longer wavelengths (which can translate into using inexpensive detection systems and low background from biological samples at these wavelengths), high extinction coefficients, relatively high quantum efficiencies, small molecular size, ease of chemical manipulation without compromising the fluorescence characteristics, and reasonable stability to reagents, pH, and temperature.
One of the major issues related to fluorescent labeling of oligonucleotides is the availability of fluorescent dyes in one or another chemical form, which would make it sufficiently user-friendly. Ideally, the chosen fluorescent tag would be available as a fully protected, modified CED-phosphoramidite. This would allow the user to simply load the labeled phosphoramidite resuspended in acetonitrile, at the extra base position (X-bottle) on a DNA synthesizer. Using the "labeling" method on the instrument would result in the direct and efficient incorporation of the label at the desired position in the oligonucleotide being synthesized. The advantage of this approach is that the number of steps involved in obtaining the labeled oligonucleotide is significantly reduced compared to indirect labeling methods. The major inconvenience is that this method implies the use of dye phosphoramidites, which are substantially more expensive and less stable, particularly to cleavage and deprotection conditions, than their standard, unmodified counterparts.
Accordingly, an indirect labeling method should be used when the chosen fluorescent tag is not available as a modified phosphoramidite. This method would still require that the fluorescent tag be presented under a form compatible with easy coupling to the synthetic oligonucleotide. The problem is not trivial, since OH groups are not very good receiving moieties in coupling reactions, and particularly since an oligonucleotide normally features two OH groups, (one at the 3' end, the other at the 5' end). In applications such as sequencing and PCR, the 5' end or any other position but the 3' end should be labeled.
For the time being, the indirect labeling method requires the incorporation of a primary amino group, most of the time at the 5' end of the oligonucleotide. This is typically achieved by adding a so-called amino-link or amino-modifier phosphoramidite as the last step in the synthesis, using the "labeling" method on the synthesizer. Some purification is needed thereafter, prior to setting up the actual coupling reaction with the fluorescent tag. The major advantage of this approach is that, once purified and 5' deblocked, the amino-oligonucleotide preparation can be further labeled according to the user's specific needs.
Typically, the fluorescent tag is added as an activated moiety, e.g., NHS-ester, to a nucleotide or oligonucleotide into which a primary amino group has been incorporated, usually at the 5' end. N-hydroxysuccinimidyl esters are well known as reactive groups, however their yields and stability in aqueous media are not often optimal. Other alternatives include the use of carbodiimides, anhydrides, and other active esters, such as paranitrophenol, to activate carboxyl groups of the fluorescent dye molecule. The less desirable aspects of existing methodologies can include a lack of selectivity of the activated product for nitrogen nucleophiles over competing species, their relative complexity to prepare (and hence their reduced cost-effectiveness), the relative lability of the activated product under coupling conditions.
For the foregoing reasons, there exists a need for novel methods to activate fluorescent dyes, for indirect methods of labeling oligonucleotides, which overcome the difficulties of the prior art. Furthermore, the method can be used to label nucleotides and oligonucleotides with a variety of fluorescent tags, including cyanine dyes, which are presently unavailable in forms suitable for direct labeling methods. Still further, there exists a need for methods of labeling nucleotides and oligonucleotides in both organic and aqueous solvents.